Effects of white mulberry powder fortification on antioxidant activity, physicochemical, microbial and sensorial properties of yogurt produced from buffalo milk

Abstract The objective of this study was to investigate the effects of sun‐dried white mulberry fruit powder (WMFP) at addition levels of 2%, 4%, and 6% for enhancing nutritional value and improving the quality of yogurt during refrigerated storage. Results showed that the highest (p < .05) antioxidant activity of 54.53 ± 0.15% was observed in 6% WMFP‐added yogurt samples. Highest pH of 4.53 ± 0.08 was observed in control. Significantly highest (p < .05) acidity (1.12 ± 0.02%) was recorded in the yogurt with 6% WMFP‐added yogurt samples on 28th day. Moreover, the addition of WMFP elevated the total soluble solids up to 20.05 ± 0.02 °Brix and water‐holding capacity up to 55.06 ± 0.34% and lessened the syneresis value (22.92 ± 0.25) in 6% yogurt samples. 2% WMFP‐added yogurt sample was given the highest sensory score in terms of overall acceptability by the panelists (8.00 ± 0.00). Thus, it was concluded that fruit powder of white mulberry can be used to improve physicochemical and sensory properties of fortified yogurt. WMFP addition to yogurt enhanced its antioxidant potential and physicochemical quality. The study introduces white mulberry‐enriched yogurt and suggests the food industries to launch this product in the market.

praised by the consumers worldwide as the most popular and indemand fermented dairy product not only because of its nutritional profile but for its health-promoting benefits as well (Savaiano & Hutkins, 2021).
Fruits, nuts, vanilla, and coffee are the flavors commonly added to yogurt. The most popular and appealed yogurt flavor among the consumers till date is strawberry flavor (Lim et al., 2019). Although dairy products have their obvious health benefits, however, fermented dairy products, such as yogurt and curd, are not documented as rich source of bioactive compounds like antioxidants and polyphenols. This necessitates to formulate the novel dairy products by supplementing and fortifying with medicinal herbs or phytochemical-rich plant extracts to satisfy the need of healthconscious consumers. In order to exploit the nutraceutical potential of mulberry, possibility of mulberry introduction as functional food and nutraceutical is need of the time by supplementation/ fortification to improve the storage stability, physicochemical, and organoleptic properties of dairy products like yogurt. To best of our knowledge, yogurt added with white mulberry has never been studies so far. Therefore, the objective of this study was to investigate the possible use of sun-dried white mulberry fruit powder (WMFP) for enhancing nutritional value and improving the quality of yogurt during refrigerated storage and evaluation of effects of WMFP on antioxidant activity, lactic acid bacterial count, physicochemical and sensory quality of yogurt.

| Sample procurement and preparation
White mulberry fruit was procured from the local market of Sargodha, Pakistan. The mulberry fruits were subjected to water washing followed by sun-drying at an ambient temperature (30°C).
The sun-dried mulberry fruits were disintegrated into small pieces and then ground using lab-scale grinder to prepare fruit powder (WMFP). Sieving with 60-mesh sieve was carried out to achieve the uniform particle size. Then, the WMFP was packed in the polythene bags, stored at freezing temperature (−18°C), and was employed in yogurt processing during storage study period.

| Proximate analysis of mulberry fruit powder
WMFP was subjected to proximate analysis as per the methods described in AOAC. WMFP was analyzed for its moisture, ash, and crude fiber through the gravimetric method as described in AOAC method No. 934.01, dry incineration in muffle furnace according to AOAC method No. 976.05 and Soxhlet method (AOAC method No. 954.02), respectively. Kjeldahl method was used for the determination of the protein content according to the AOAC method No.
976.05. The obtained results were indicative of the total nitrogen content which were further multiplied by the factor 6.25 for total protein content determination. Difference method was employed for the determination of the total carbohydrate content as per the following Equation 1: TPC and TFC of WMFP were determined spectrophotometrically at 765 and 510 nm, respectively, according to the methods described by Jiang et al. (2021). The TPC was expressed as gallic acid equivalents (mg GAE 100 g −1 ) on dry weight (DW) basis, whereas TFC results were represented as milligram rutin equivalent (mg RE 100 g −1 ) on DW basis. yogurt samples were kept at 5°C for 28 days. All the experiments were carried out in triplicate (n = 3).

| Determination of antioxidant activity
Antioxidant potential of WMFP-added yogurt samples was measured according to the method described by Blois, 1958. Homogenization of 1 g yogurt with 10 ml of ethanol was done. 0.2 ml of the mentioned solution was mixed with 0.8 ml of DPPH solution (1.5 × 10 −4 M) and was allowed to stand for 30 min at a place where no light could approach it. Absorbance was measured by UV spectrophotometer (UV-1800; Shimadzu Instruments Mfg. Co., Ltd.) at 517 nm against a blank. All measurements were performed in triplicate. The degree of discoloration was measured by the following equation: Abs control represents the absorbance of DPPH radical with ethanol and Abs sample symbolizes the absorbance of DPPH radical with yogurt samples.

| Physicochemical analysis
2.5.1 | pH and acidity pH meter (pH 211 Microprocessor pH meter; Hanna Instruments) was used to measure the pH values of the samples. Yogurt was homogenized in water (1:9 ratio) preceding to pH determination as per AOAC method 943.02 (AOAC, 2000). The titratable acidity was determined by titration using a 0.1 N sodium hydroxide (NaOH) solution to the endpoint of pink color.

| Total soluble solids
Yogurt (2 g) was placed in a hot air oven for 3 h at 100°C and then cooled in a desiccator for 30 min. The percentage of residues obtained was the total soluble solids (TSS) (AOAC, 2000).

| Water-holding capacity (WHC)
The centrifugation of 20 g yogurt sample at 669× g for 10 min was carried out and the supernatant was removed and weighed (Arslan & Ozel, 2012).

| Syneresis
A 200 g of each of yogurt samples (Y) was taken and centrifuged at 4°C for 10 min at 770× g. The syneresis was measured as per the reported method of Abd El-Salam et al. (1991).

| Lactic acid bacteria count
Yogurt samples were diluted by serial dilution with peptone water.

| Sensory evaluation
Sensory evaluation was carried out by the 20 semi-trained panelists in laboratory using a 9-point hedonic scale with following score expressions: 9 = like very much, 8 = like a lot, 7 = rather like, 6 = quite like, 5 = neither like nor dislike, 4 = dislike a little, 3 = rather dislike, 2 = dislike very much, 1 = highly dislike. Different sensory attributes were evaluated, such as color, flavor, texture, sweetness, sourness, taste, and overall acceptance. All individuals were instructed well before analysis. All measurement were recorded in triplicate (n = 3).

| Statistical analysis
Results obtained from different parameters were subjected to statistical analysis using one-way analysis of variance (ANOVA) technique. The differences between the means were determined through Duncan multiple range test at significance level of p < .05 (Steel, 1997).

| RE SULTS AND D ISCUSS I ON
Nutritional composition (protein, fat, crude fiber, ash, and carbohydrate) in terms of g/100 g on dry weight (DW) basis, total phenolic content (TPC: mg GAE/g DW), antioxidant activity (AOA %), and flavonoid content of WMFP is provided in     flavonoid content were found to be 13.82 mg GAE/g DW, 31.96%, and 4.72 mg/g (DW basis), respectively.

| Effect on antioxidant activity (DPPH %) of yogurt
The results of effect of WMP concentration and storage period on antioxidant activity (DPPH %) of yogurt are given in Table 2.  (Devi et al., 2013). The increase during storage may be attributed to the metabolic activity of LAB that remains active during refrigerated storage (Hammon & Blum, 1998). Increased antioxidant activity in fermented milk products is due to the proteolysis by lactic acid bacteria and the subsequent release of bioactive peptides (Pihlanto, 2006). These results were in agreement with the findings of Sung et al. (2015) who reported increases in antioxidant activity of fortified yogurt because of the presence of tyrosine amino acid in fortified yogurt composition as the amino acid tyrosine has been reported to exhibit phenolic side chain in its configuration (Chen et al., 2006). Moreover, the highest antioxidant potential of supplemented yogurts might be attributable to the increased anthocyanin content as anthocyanins are group of compounds which have contributory role in imparting of color to the fruits, plants, and flowers (Thompson et al., 2007). During storage, the 6% supplemented yogurt samples did not exhibit any significant decrease in antioxidant activity which implied that antioxidant compounds in yogurt remained stable with slight changes in decreasing manner owing to anthocyanins stability in supplemented yogurt samples as well as compared to control. Moreover, the higher antioxidant activity of supplemented yogurt samples was attributed to the individual M.
alba phytochemical contents and microbial metabolic activities during growth phases of mulberry plant (Thompson et al., 2007). During refrigerated stoppage, the decreasing trend in antioxidant activity of yogurt samples could be ascribed to the fact of phenolic compounds TA B L E 1 Nutritional composition (g/100 g DW), TPC (total phenolic content mg GAE/g DW), and antioxidant activity (AOA %) of white mulberry

TA B L E 2
Effect of white mulberry powder concentration and storage period on antioxidant activity (DPPH %) of yogurt degradation or increased formation of complex by interaction of polyphenols with milk proteins (Yuksel et al., 2010).

| Effect on pH, titratable acidity, and TSS of yogurt samples
The results of WMFP-supplemented and control yogurt samples are given in Table 3 during storage period ranging from 1 to 28 days. As evident from results of  (Sung et al., 2015). Acid development in slow manner is undesirable property for yogurt development; therefore, increased acidity in supplemented yogurts was evident of the fact that WMFP caused stimulation of whey separation and symbiotic relationship breakdown among started bacteria (Celik & Bakirci, 2003). The decrease in pH is indicative of rises in acidity levels, and hence, the results corroborated with the decreasing pH tendencies with corresponding rises in storage period and WMFP addition levels from 2% to 6%. As evident from results of control, the titratable acidity levels on day 1, 7, 14, and 28 were found to be in ranges of 0.90%-0.98%, 0.92%-0.99%, 0.95%-1.03%, 0.102%-1.08%, and 1.05%-1.12%, respectively. In a similar study conducted by Lee and Lee (2014), pH was found that 20% supplemented yogurt samples with barley flour exhibited higher acidity values as compared to that of control (un-supplemented) samples. Titratable acidity of yogurt samples enhanced owing to the presence of organic acids in composition of white mulberry fruit powder. In this regard, notable organic acids possibly may include butyric, acetic, formic, citric, and lactic acids. These results are in agreement to the previous studies where authors suggested that white mulberry has the general effect to lessen the pH of yogurt (Mahmood et al., 2008;Tarakci et al., 2013). The increase in acidity may be attributed to the growth of lactic acid bacteria (Servili et al., 2011).
As evident from results of

| Effect on water-holding capacity and syneresis
All WMFP-supplemented (2%, 4%, and 6%) and control yogurt samples were evaluated in terms of their WHCs and syneresis. The results of WHC of yogurt samples are tabulated in Table 3. As evident from results of  Table 4). One of the most influential factors is degree of denaturation of whey proteins which exert significant effect on yogurt texture and syneresis. Furthermore, incubation temperature also plays its role in terms of affecting syneresis. In this regard, lower temperature allows to take longer time for setting of curd whereas gradually increasing ten- The decreasing trend in syneresis might be correlated to the fact of rises in WHC of mulberry fruit powder (Öztürk et al., 2018). This addition might have attributed to enhance the available carbohydrates, fiber, and pectic substances having high water-binding ability (Singh & Muthukun, 2008).

| Effect on lactic acid bacterial count
The lactic acid bacteria (LABs) count (S. thermophilus and L. bulgaricus) during storage interval ranging from 1 to 28 days for control and WMFP-supplemented samples are demonstrated in Figure 1.  (Figure 1).
Addition of white mulberry significantly (p < .05) increased the viable counts of S. thermophilus and L. bulgaricus. This increase might be attributed to the presence of polyphenols and fiber in food of a plant origin. Dietary fiber provided additional sources of carbohydrates and acts as fermentable substrate favoring lactic acid bacteria growth. Commonly, the viable counts of Streptococcus in yogurt is significantly greater than that of Lactobacillus (Zacarchenco & Massaguer-Roig, 2006). A decrease in the viable count of yogurt bacteria during storage was reported by Cais-Sokolińska and Pikul (2004). The yogurt sample added with 2% WMFP had significantly higher sensory values as compared to other samples ( Table 5).
The LAB count was considerably higher at start of the storage In a study carried out by Shokryazdan et al. (2015), about 38 LABs were isolated from the mulberry silage; however, only four strains were capable enough for survival in GIT tract. Mulberry pekmez has been reported to exhibit potential of lowering LAB count because of rises in the TSS and sugar content, and hence, it may be implied that LABs growth was significantly affected with corresponding rises in additive levels of mulberry fruit powder in fortified yogurts (Celik & Bakirci, 2003).

| Effect on sensory attributes of supplemented yogurt
Sensory evaluation allows to record the preferences of human beings in terms of scores related to various sensory attributes, such as taste, texture, appearance, etc. Sensory evaluation was carried out for two types of yogurt samples, such as control and WMFP-added yogurt samples (2%, 4%, and 6% supplemented samples). The color values for control samples during all storage periods ranged 5.33-7.01, whereas, for 2%, 4%, and 6% supplemented yogurt samples, the color values were observed in ranges of 6.01-7.67, 5.01-6.67, and 4.67-6.01, respectively. As evident from results of WMFP-supplemented samples (T 1 ) subjected to storage period from 1 to 28 days was found in range of 5.34-7.01. In case of 4% WMFP-supplemented yogurt samples, the flavor was found to be in range of 5.01-6.34 during all storage intervals spanning 1-28 days.
In case of 6% WMFP-supplemented yogurt samples, the flavor was

| CON CLUS IONS
White mulberry has the potential to help in curing of diabetes mellitus, atherosclerosis, cardiovascular diseases, different neurological disorders, various skin infections as well as cancer. To best of our knowledge, yogurt added with white mulberry has never been studies so far. Therefore, the objective of this study was to investigate the effect of sun-dried white mulberry fruit powder (WMFP) (2%, 4%, and 6%) for enhancing nutritional value and improving the quality of yogurt during refrigerated storage and evaluation of effects of white mulberry fruit powder (WMFP) on antioxidant activity, lactic acid bacterial count, physicochemical, and sensory attributes of yogurt. Results showed that the highest (p < .05) antioxidant activity of 54.53 ± 0.15% was observed in 6% WMFP-added yogurt samples.
Highest pH of 4.53 ± 0.08 was observed in control. Significantly highest (p < .05) acidity (1.12 ± 0.02%) was recorded in the yogurt with 6% WMFP-added yogurt samples on 28th day. Moreover, the addition of WMFP elevated the total solids up to 20.05 ± 0.02 °Brix and water-holding capacity up to 55.06 ± 0.34% and lessened the syneresis value (22.92 ± 0.25) in 6% yogurt samples. 2% WMFPadded yogurt sample was assigned the highest sensory score in terms of overall acceptability by the panelists (8.00 ± 0.00). The results demonstrated that the addition of WMFP significantly (p < .05) improved the antioxidant potential and quality of yogurt. Thus, it was concluded that fruit powder of white mulberry can be used to enhance antioxidant potential, physicochemical, and sensory properties of fortified yogurt. This addition in yogurt enhanced its antioxidant potential and physicochemical quality. The study introduces white mulberry-enriched yogurt and suggests the food industries to launch this product in the market. Therefore, white mulberry fruit powder can be incorporated in yogurt for elevating nutritional value, improving yogurt quality, and for enhancement of yogurt functionality.

ACK N OWLED G M ENT
There was no funding received for this study from any funding organization.

CO N FLI C T O F I NTE R E S T
Authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.